Cryo-EM structures of the translocational binary toxin complex CDTa-bound CDTb-pore from Clostridioides difficile

Some bacteria express a binary toxin translocation system, consisting of an enzymatic subunit and translocation pore, that delivers enzymes into host cells through endocytosis. The most clinically important bacterium with such a system is Clostridioides difficile (formerly Clostridium). The CDTa and CDTb proteins from its system represent important therapeutic targets. CDTb has been proposed to be a di-heptamer, but its physiological heptameric structure has not yet been reported. Here, we report the cryo-EM structure of CDTa bound to the CDTb-pore, which reveals that CDTa binding induces partial unfolding and tilting of the first CDTa α-helix. In the CDTb-pore, an NSS-loop exists in ‘in’ and ‘out’ conformations, suggesting its involvement in substrate translocation. Finally, 3D variability analysis revealed CDTa movements from a folded to an unfolded state. These dynamic structural information provide insights into drug design against hypervirulent C. difficile strains.

the loop and locking in one conformation. Does this change the toxicity of CTD or does it change the CDTa/CDTb affinity? Moreover, the authors build both loops in a 0.5:0.5 occupancy into the map. Are more focused refinements, sortings, eventually combined with signal subtraction possible to sort out specific conformations at the NSS loops? If the authors performed this without success, it should be stated in the manuscript.
Is a difference between the NSS loops in class 1 (with D4II bound to the beta-barrel stem) and in class 2 (D4II not resolved) eminent? Already outward-fixed NSS loops with stem contacts as depicted in Fig. 6, left panel, would be a disagreement between the scheme in Fig. 6 and the data. I recommend to rename "classes 1 and 2" according to their features, e.g., class 1 as long-stem class. This would make reading more straightforward. The interpretation of CDTa binding and translocational unfolding dependent of the NSS loop switch is explained with class 2 only. What can additionally be interpreted with class 1? The unique findings in class1 are not described and interpreted -contacts between domain 4 with the beta-barrel stem, as depicted in the schematic in Fig. 6.
Further points to address: Abstract: The authors start with TcdA and TcdB, two C. difficile toxins that are not part of the work. I recommend to remove this distracting part and instead start with CDTa/CDTb as toxin translocation system. l. 28: grammatical error (remove "a") Introduction: l. 46f: "Additionally, some bacteria have a unique translocation system named binary toxin": Can the system only translocate toxins or also other proteins? If the first is the case, I would define it as toxin translocation system. l. 59: indicate a citation for "internalized by receptor-mediated endocytosis". l. 68 and 70: Is anthrax PA the "second" group? I recommend to write "second" instead of "another" in l. 68.
l. 74 -86: I recommend to put the general chapter of C. difficile before the detailed description of binary toxin systems. The chapter here appears to be out of context. l. 133 ff: As it is described here, LMNG was added after cleavage? Can LMNG during cleavage completely stop the formation of heptamer dimers, or eventually higher concentrations of the detergent? I recommend to show more data here as SI data. No biochemical and biophysical data at all are shown here (negative staining, SDS gels, …).
l. 137/138: There is a sudden transition from sample preparation to cryo-EM 3D classification, a sentence describing electron microscopy would be helpful for understanding. l. 140f: percentages instead of ratios of classes would be more intuitive.
l. 149: Write and interpret only one digit for cryo-EM structures.
l. 159: I recommend to re-phrase "seemed to contain one whole beta-barrel stem", since it is not complete and therefore distracting. l. 181: di-calcium site: Is there a difference in the seven subunits? As it is described in methods, no symmetry was applied. Are there Ca2+ binding differences with respect to CDTa binding? l. 186: stable region -do you mean conserved region or physically stable? l. 188: His 314 -please indicate in the seq alignment in SI Fig. 1 and describe exactly in which family the residue is conserved. l. 192ff: Is the position of CDTa identical to the prepore state and to the position of Ia in Iota toxin? Are there other differences to the crystal structure than in the N-terminal helix? Is the N-terminal alpha helix artificially stabilized in the crystal structures of CDTa and Ia by contacts? If this is the case, no destabilization by CDTb would occur. If this is the case, please also check the corresponding section in the discussion.
l. 202ff, loop conformations: please indicate which residues were built in two conformations. Is the map/model fit and the geometric properties identically good in both conformations? Is the occupancy 0.5 and 0.5? Please also see my comment above (3D sorting, additional experiments). l. 206: Please describe your re-analysis in the Methods section, and refer to SI figure 5 here, too. Since here C7 symmetry was applied, differences between subunits are not determinable. Does the 3.2 Å double pore structure obtained in this work show differences? l. 210: "Thus, we conclude that the NSS-loop conformation is in equilibrium between the two states in the default pore structure" -in the sentences above, it is described that both conformations exist both in the prepore and in the pore part. Please double-check and re-phrase unambiguously.
l. 214ff: I recommend to introduce a new chapter heading for CDTa. How "deeply" does CDTa penetrate into the pores? l. 219: Is density at low binarization threshold and/or after lowpass filtering visible that corresponds to the missing residues? In Methods, it is described that CDTa starts at residue 53 -please explain this discrepancy and re-number residues. l. 220: Is the "steric hindrance" part of the mechanism? Here, it can be interpreted that it is nonphysiological. From an unbiased point of view, I would expect that the missing residues are stabilized by the contacts with CDTb. Are data available that an N-terminally truncated CDTa variant binds to CDTb and/or is translocated? If not, I suggest an additional experiment to confirm this. Discussion: l. 233: Does a mutation of F774L in CDTb also disvavor heptamer dimerization? If no data are available, this would be a quick experiment to perform. l. 237f: Although the di-heptamer might be protected from proteolysis, does it have any physiological relevance? Is di-heptamerization reversible? Since the authors suggest a concentration-dependent behavior, is it possible to confirm this and find out the K(D)?
l. 247: This would make the NSS loop to a NSX loop. l. 249: use "a"instead of "the" l. 257f: Please describe in the methods the model re-building of Ib. Can the "weaker" out-conformation density than in-conformation density be described in a quantitative manner? In addition, the fact that two different loops are locked in the out state in Ib is not discussed at all. What are possible causes or consequences? l. 261: "not so strong" -how strong does the binding need to be? I would estimate that the K(d) has to be in the range of physiological concentrations. Are there affinity constants available for CDT or Iota or other, comparable toxins of this family? l. 265ff, anthrax PA: A SI figure that compares the dimensions of CDT/Iota and anthrax PA would be helpful.
l. 275: Where is the alpha clamp in CDTb and Iota, this has not been stated in the manuscript before. Is it the C-alpha edge? l. 280: the N-terminal helix of CDTa and Ia? l. 314f: The open states were only observed for Anthrax PA, which has likely another mechanism. Are any indications for a phi-clamp opening available in CDT or Iota toxins? l. 317: What did the cited study of the PA translocation complex show? Is it relevant for the CDT translocation mechanism? l. 322: Can in silico docking experiments reveal how the described inhibitors could block translocation?
Methods: Some issues (missing description of re-modelling of two structures) were already described in the corresponding results section.
Sample preparation: Can LMNG already be added for proteolysis? Does this inhibit heptamer dimerization completely (or higher conc. Of LMNG)? Has unbound CDTa been removed for cryo-EM?
Cryo-EM data collection: At which concentration was the protein vitrified?
Data processing: Has C7 symmetry been applied for any step of data processing? Please also show the initial model of cryosparc that was used for the initial 3D sorting in SI Fig. 2. At which pixel size were the initial steps carried out (until re-extraction)?
l.404: Alternating CTF refinements and 3D refinements? What resolution improvement did this step yield? l.406: Please describe the "non-align 3D classification" of CDTa in more detail -were all particles used for this part afterwards? Were misoriented particles rotated into an orientation that CDTa fits?        In their manuscript "Cryo-EM structures of the translocational binary toxin complex CDTa-bound CDTbpore from Clostridioides difficile", Kawamoto and coworkers present a structure model for the protein complex formed by the two components of the binary toxin CDT from Clostridioides difficile, which is based on detailed cryo-EM analysis. Moreover, they provide a more detailed model for the binding of the enzyme subunit CDTa to the hepatmeric binding subunit CDTb and the insertion and translocation of partially unfolded CDTa into and through the CDTb pore.
The results of this study confirm and extend the knowledge about the structure and function of CTa/CDTb, which was obtianed in recent years by other groups, also using cryo-EM as well as urther biophysical approaches such as X-ray crystallography and NMR. These former studies revealed a CDTa/CDTb complex with a heptameric structure for CDTb, too, and they also proposed a model for the interaction of CDTb with CDTa and the translocation of CDTa via CDTb. However, in these earlier studies, CDTb was found to be a di-heptamer, while the present work, most likely due to its novel purification protocol for the CDTb pore and a higher resolution, demonstrates that in addition to di-heptamers, CDTb also exisits as a single heptamer in the CDTa-bound CDTb pores. This is a central new finding of this manuscript. The authors suggest in their discussion that this is the biologically active CDT complex, which exists in low CDTb concentrations and exhibits toxic effects on cells by delivering CDTa, while CDTb di-heptamers might exist under high CDTb concentrations to prevent aggregation and/or degradation of this protein.
Based on cryo-EM, the authors performed a detailed analysis of CDTb heptamers and their interaction with CDTa. They found four crucial elements of the CDTb heptamers, i.e. calcium edges that serve for binding of CDTa, NSS-loops that are also involved in CDTa binding and likely crucial for CDTa unfolding and insertion into CDTb pores, a Phe-clamp, which was earlier found in PA63 and Ib heptamers from the related binary anthrax and iota toxins, too, and a stem which forms the channel below the Phe-clamp.
Their results revealed that CDTa has a N-terminal and C-termimal domain and the first two alpha-helices of the N-terminal domain partially unfold after binding to CDTb pores and 9 amino acid residues within the first helix penetrate deeply into the pore and induce the unfolding of CDTa. Here, the NSS loops of CDTb are involved by switching between an "in" and "out" conformation, which makes the strong binding of CDTa to CDT b more loosely and enables translocation into the channel of CDTb pores.
Here, it would be very interesting to investigate the CDTb/CDTa interaction under acidic conditions, as in endosomes where CDTa translocation through CDTb pores occurs in living cells. This could be included in the manuscript in direct comparison to the interaction under neutral pH conditions. We thank three reviewers for their careful reading and helpful comments. Please find below our point-by-point response. In the revised manuscript, we added two new biochemical data: One is mutational studies of F774 (receptor binding domain's key residue) to see how important this residue is for the di-heptamer formation. The second one is the CDTa-CDTb-pore binding studies of wt and two mutants of NSS-loop (NSS>SPS and NSS>PSS) by surface plasmon laser. Furthermore, a new 3D variability analysis of short CDTa-bound CDTb-pore showed CDTa movements from a folded to an unfolded state. We hope that these new data strengthen our findings and the paper will be acceptable in Nature communications.

Reviewer #1
In summary, this work presents a very nice description of the CDTb-CDTa interaction for a monomeric heptamers (pore-state) at a higher resolution that previously. Details in these structures revealed the dynamic conformation (and potential role) of the NSS-loops for the binding of CDTa, previously missed in the electron density maps of previously resolved complexes. Also, it provided further details on the location of the N-terminal alpha-helix 1, that lead the authors to hypothesize a steric induced unfolding of the Nterminus of the helix, that may be required to initiate translocation. All the conclusions made in this work are from the observation and analysis of the obtained structure/s, no accompanied experimental data to support the proposed hypothesis was provided (not unusual for some structural research). Despite the highest resolution description of the complex, the slight differences found between the presented and previous published structures reduce the enthusiasm about the work. > Thank you for your comments. We provided further details of N-terminal -helix 1, and this discussion was further studied by 3D variability analysis, which presents CDTa motion in the complex. We added the motion picture of the N-terminal -helix unfolding to thread it to the -clamp (Supplementary Video). Furthermore, we added two new biochemical data: One is mutational studies of F774 (receptor binding domain's key residue) to see how important this residue is for the di-heptamer formation (new Fig.1).
The second one is the CDTa-CDTb-pore binding studies of wt and two mutants of NSSloop (NSS>SPS and NSS>PSS) by surface plasmon laser (new Table 2 and new Fig. S4).
We hope these data support the first details of the unfolding mechanism of CDTa via CDTb-pore.

Reviewer #2
With the additional information obtained in the present work due to the so far unmatched resolution, the authors re-analyzed the Iota toxin structure and identified a similar conformational switch in the NSS loop like here. Therefore, the present work extends the conceptual understanding of this class of toxins and is suitable for publication in Nat Comm. > Thank you for your valuable comments. We sought to respond as much as we can and provide two additional biochemical data.
Moreover, the authors build both loops in a 0.5:0.5 occupancy into the map. Are more focused refinements, sortings, eventually combined with signal subtraction possible to sort out specific conformations at the NSS loops? If the authors performed this without success, it should be stated in the manuscript.
> We tried the more focused refinements, but it did not succeed in differentiating the two states (in and out states of NSS-loop) clearly. To see the variability, we tried the 3D variability analysis using cryoSPARC2. In the input, we feed all particles from relion's analysis of short CDTa-CDTb-pore (new Fig.S2 and new Fig.6). Then, using a series of maps, we generated one movie (Supplementary Video) and added two refined structures, the folded CDTa complex and the unfolded CDTa complex.
Though we could not evaluate the accurate occupancy of NSS-loop, we found differences of the NSS-loop conformations upon the folded and the unfold CDTa complex by the 3DVA analysis. We added as follows " Two NSS-loop conformations would adapt to fit transient translocational CDTa states: the unfolded and folded complexes showed different 'in' and 'out' conformations in the subunit D NSS-loop (Fig. 6h,i). The short CDTa-bound CDTb-pore map represents the total map, showing the small differences between folded and unfolded CDTa (Fig.   4a,b)." (L239-243) We believe these are very exciting data to support our discussion of the initial unfolding process of CDTa via CDTb.

>
In the long map, we also observed not only the unfolding and tilting of the first Nterminal -helix of CDTa but also the same two states of NSS-loop as observed in the short map. However, the short map quality is better than the long, especially in CDTa.
Thus, we went in the next step by the 3DVA using in the short map.
The unique findings in class1(long) are not described and interpreted ＞ We added the relationship between domain D4II and the stem formation in the second paragraph of discussion. "The long and short structures, with other reported structures, of CDTb, including di-heptamers, are summarised in Supplementary Fig. S5. Between the long and the short structures, the definitive difference is that D4II is clearly visible in the long form. That is, after stem formation, D4II is stably fixed around the stem. In di-heptamer structures, two different conformations of D4II were observed. These dynamic features of D4II are likely important in binding the host cell LSR. In our previous study, we confirmed that the receptor-binding domain is also involved in determining oligomerization efficiency, using a D4II-deficient mutant Ib 1 ." Further points to address: Abstract: The authors start with TcdA and TcdB, two C. difficile toxins that are not part of the work. I recommend to remove this distracting part and instead start with CDTa/CDTb as toxin translocation system. l. 28: grammatical error (remove "a") >Thank you for your comment. I revised the first part of the abstract.
(we deleted the TcdA and TcdB parts from abstract.)    .1a). There was no difference of di-heptamers when LMNG added before chymotrypsin treatment (Fig 1b). There was also no difference of the ratio of diheptamer and heptamer formation when 5 times LMNG added (data not shown).
Furthermore, we showed new data of F774L and F774G: F774 is the critical residue for the di-heptamer formation by the density gradient centrifugation method (Fig 1e  and f). Because I would like to show that the short is higher resolution structure (substantially, the map is better than long in CDTa part), thus I would like to keep it.
(17) l. 159: I recommend to re-phrase "seemed to contain one whole beta-barrel stem", since it is not complete and therefore distracting. > OK. We changed as follows.
"The former contained one entire -barrel stem; however, the final map calculation showed that the tip of this stem (residues 337-358) had very weak density; therefore, we modelled the structure with a partial -barrel stem, excluding residues 337-358 ( Supplementary Fig.S2d l. 163: Not only class 2, but also class 1 do not contain the whole beta-barrel stem.
>As we described in the method, the long have some visible map of whole stem. However, it is not possible to put the whole stem model because of the weak density.
We added the TM part in Fig.2. Also, we showed the difference of the stem between long and short in Supplementary Fig. S2d.   >Yes. We deleted one sentence and just explained as follows and showed in Fig. 3e and   3f. " The lumen of the pore contained four constrictions. "(L188) (24) l. 181: di-calcium site: Is there a difference in the seven subunits? >No. As it is described in methods, no symmetry was applied.
Are there Ca2+ binding differences with respect to CDTa binding? > There are no differences in the di-calcium site.  > Though we did not refine the coordinate, we confirmed two states in the diheptamer by visual inspection of the map. We added in the legend of Fig.S6 as follows. "CDTb di-heptamer cryo-EM map (EMD-20926) and Ib-pore map (EMD-0721) were Since here C7 symmetry was applied, differences between subunits are not determinable. Does the 3.2 A double pore structure obtained in this work show differences?
> We obtained the maps of CDTa-bound di-heptamer. Even we applied C1 symmetry calculation, CDTa and the binding site in CDTb were averaged. In this meaning, we did not model of the structure.
(30) l. 210: " Thus, we conclude that the NSS-loop conformation is in equilibrium between the two states in the default pore structure" -in the sentences above, it is described that both conformations exist both in the prepore and in the pore part.
Please double-check and re-phrase unambiguously. > Thank you. As shown in the map new SI fig. 6a and b, both conformations exist both in the prepore and in the pore part. Thus, we revised as follows " Thus, we conclude that the NSS-loop conformation is in equilibrium between states in the default pre-pore and pore structures." L218~220 (31) l. 214ff: I recommend to introduce a new chapter heading for CDTa. > I changed the name of the chapter "CDTa-binding mode and translocational unfolding ".
How "deeply" does CDTa penetrate into the pores? > I deleted "deeply". In Methods, it is described that CDTa starts at residue 51 -please explain this discrepancy and re-number residues.
> It includes signal peptide. In Ia case, the signal peptide was shown in Popoff paper > We find that di-heptamer and heptamer could be separated by cryo-EM classification and also by ultracentrifugation. We tried blue-native page, but it results that di-heptamer break to single heptamer (data not shown). From this, we could not evaluate whether it is a concentration-dependent behavior.
Though it it not sure that the di-heptamer is physiologically important, it is sure that di-heptamer can not stick into lipid bilayer. I added the results as follows " When WT is bound to LSR via Can the " weaker " out-conformation density than in-conformation density be described in a quantitative manner?
>No. But there are clear difference between in and out of visible density.
In addition, the fact that two different loops are locked in the out state in Ib is not discussed at all. What are possible causes or consequences? > We can provide clear explanation of CDTb in the discussion with 3DVA analysis ( Supplementary Fig. S7). In Ib case, we could explain subunit F : "out" is caused by steric hindrance like CDTb. However, in subunit E, we could not explain well at present.

Sample preparation:
Can LMNG already be added for proteolysis? > No, we do not add for proteolysis. (This is standard protocol as described in new Fig.1a) Furthermore, we checked whether the timing of LMNG (before and after protease treatment) would affect the di-heptamer or heptamer formations (new Fig.1b,c). But we did not observe any difference of the di-heptamer or heptamer formations. We also showed the two mutants F774L and F774G decreased the di-heptamer fraction (new >No, we did not inhibit the di-heptamer formation with five times more LMNG (data not shown ).
Has unbound CDTa been removed for cryo-EM? >No, we did not . This is the same protocol as iota-toxin Ia-bound Ib-pore structural determination 6 .
Thus, we added the method.
(48) Cryo-EM data collection: At which concentration was the protein vitrified? >We added the method. (L428) Data processing: Has C7 symmetry been applied for any step of data processing? >No, we did not.
Please also show the initial model of cryosparc that was used for the initial 3D sorting in   However, as we described before, from 3DVA, we refined two more coordinates for folded CDTa and unfolded CDTa complex. In the folded and unfolded CDTa complex, we built CDTa with whole N-terminal residues as described in method.  > We revised as the new Fig.7, which summarize the scheme from complex formation to translocation.
(57) Are the 2ndary structures in b from the crystal structures or from the structure here? >Those are from cryo-EM structure.
4. Please also state which ones were processed with C7 symmetry.
We did not use C7 symmetry.

Reviewer #3 (Remarks to the Author):
The results of this study confirm and extend the knowledge about the structure and function of CTa/CDTb, which was obtianed in recent years by other groups, also using cryo-EM as well as further biophysical approaches such as X-ray crystallography and NMR. These former studies revealed a CDTa/CDTb complex with a heptameric structure for CDTb, too, and they also proposed a model for the interaction of CDTb with CDTa and the translocation of CDTa via CDTb. However, in these earlier studies, CDTb was found to be a di-heptamer, while the present work, most likely due to its novel purification protocol for the CDTb pore and a higher resolution, demonstrates that in addition to di-heptamers, CDTb also exisits as a single heptamer in the CDTa-bound CDTb pores. This is a central new finding of this manuscript. The authors suggest in their discussion that this is the biologically active CDT complex, which exists in low CDTb concentrations and exhibits toxic effects on cells by delivering CDTa, while CDTb di-heptamers might exist under high CDTb concentrations to prevent aggregation and/or degradation of this protein.
Based on cryo-EM, the authors performed a detailed analysis of CDTb heptamers and their interaction with CDTa. They found four crucial elements of the CDTb heptamers, i.e. calcium edges that serve for binding of CDTa, NSS-loops that are also involved in CDTa binding and likely crucial for CDTa unfolding and insertion into CDTb pores, a Phe-clamp, which was earlier found in PA63 and Ib heptamers from the related binary anthrax and iota toxins, too, and a stem which forms the channel below the Phe-clamp.
Here, it would be very interesting to investigate the CDTb/CDTa interaction under acidic conditions, as in endosomes where CDTa translocation through CDTb pores occurs in living cells. This could be included in the manuscript in direct comparison to the interaction under neutral pH conditions. > Thank you for the comments. We added new SPR experiments in the manuscript and presented the CDTa/CDTb interactions under acidic conditions as well as neutral condition. Furthermore, we also the CDTa/CDTb interactions WT (NSS-loop) and two NSS-loop mutants. Though we did not expect, under acidic conditions, the interaction of CDTa with CDTb-pore did not decrease. We consider it necessary to maintain the interaction before the translocation, even at the acidic pH.
Furthermore, we added two new coordinates folded CDTa and unfolded CDTa from new 3D variability analysis results. It provides one supplementary video which supports the initial unfolding of CDTa in CDTb-pore. We believe these are exciting results.
The authors have addressed my comments and suggestions in an appropriate way. Especially the rearrangement of the figures makes the manuscript more accessible. The additional data, in particular the 3D variability analysis that resulted in a clear description of the initiation of the translocation mechanism by the N-terminal CDTa helix, clearly improve the manuscript and support the author's hypothesis. Therefore, I recommend publication of the revised version.
Minor issues to address: SI Fig. 4: It would be helpful to label the SPR curves for the individual events (association/dissociation), and also plot the fits into the data curves. Also, reference to Table 2 (and from table to   Reviewer #2 (Remarks to the Author): The authors have addressed my comments and suggestions in an appropriate way.
Especially the rearrangement of the figures makes the manuscript more accessible. The additional data, in particular the 3D variability analysis that resulted in a clear description of the initiation of the translocation mechanism by the N-terminal CDTa helix, clearly improve the manuscript and support the author's hypothesis. Therefore, I recommend publication of the revised version.
Minor issues to address: (1) SI Fig. 4: It would be helpful to label the SPR curves for the individual events (association/dissociation), and also plot the fits into the data curves. Also, reference to  Fig.4 as the reviewer suggested, adding the plot the fits and triangles indicate the start of association and dissociation, respectively.
We also added the reference in legend in Table2.